Isoforms of the human sst5 receptor originated by alternative splicing and oligonucleotide pairs to detect them by pcr

ABSTRACT

The present invention is referred to two human nucleic 5 acids that comprise sequences encoding two new isoforms of the human somatostatin receptor type 5 originated by alternative splicing, named sst5B and sst5C, with possible involvement in tumor processes. In addition, the invention is referred to oligonucleotide pairs allowing 10 their differential detection in several tissues using the PCR technique.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/713,058, filed Dec. 13, 2012, pending, which is a division of U.S.application Ser. No. 12/738,131, filed Apr. 15, 2010, now U.S. Pat. No.8,354,273, issued Jan 15, 2013, which is a United States national stagefiling under 35 U.S.C. §371 of international (PCT) application no.PCT/ES2007/000627, filed Oct. 17, 2007, and designating the U.S.

PURPOSE OF THE INVENTION

The present invention refers to two new isoforms of human somatostatinreceptor type 5, as well as their detection in biological samples.

BACKGROUND OF THE INVENTION

The hypothalamic neuropeptide somatostatin (SRIF) acts in a multitude oforgans and target tissues throughout the body, fundamentally exercisinga inhibiting effect, whether over the secretion and regulation of otherhormones or other diverse biological processes (Moller et al., 2003).

This action, which is generally inhibiting, but on some occasionsstimulating, (Castaño et al., 1996) is exercised through a family ofseven transmembrane domain (7TMD) type receptors coupled to G-proteins(GPCR), called somatostatin or ssts receptors. All ssts share a commonstructure consisting of an extracellular amino-terminal end connected toseven hydrophobic domains inserted into the membrane, which are in turnjoined together through eight hydrophilic domains, and they end in anintracellular carboxyl-terminal end, which is important for themodulation of second messenger routes.

Up to the present, there are five different subtypes of ssts in mammals,from sst1 to sst5, and there are also two isoforms from subtype 2 (sst2Aand sst2B) in mice and rats produced through alternative adjustment ofthe precursor messenger RNA which code two different proteins in theintracellular carboxyl-terminal region which present differentproperties with regard to the modulation of intracellular signalingroutes. In fish, however, other isoforms from each receptor subtype havebeen discovered due to gene duplication phenomenon in place of otherdifferential adjustment phenomenon of the messenger RNA that codes eachof the isoforms.

The GPCRs, including the ssts, are involved in numerous medicallyrelevant cellular processes, measured by signal transduction routesthrough G proteins. More specifically, one of these ssts subtypes, thehuman sst5 (WO 0177172, WO 0155319, WO 0136446, EP 1369698, WO 03104816)has been linked to a multitude of diverse pathologies in mammals, suchas hematological and cardiovascular diseases, central and peripheralnervous system disorders, cancer, inflammatory processes, hepaticdiseases, gastrointestinal and genito-urinary diseases (WO 03104816).

The human somatostatin receptor type 5, sst5, is recorded in public databases with access numbers, including but not limited to GI39756975,GI21954086, GI13937340, which contain sequences pertaining to the DNAcopy of the coding sequence, as well as genomic sequences that containthe complete gene structure of said receptor, and in addition to thecoding sequence, the promoter region contains intronic sequences anduncoded 5′ and 3′ regions. As of the present, no alternative processingof human sst5 messenger RNA has been described which brings about anydifferent alternative isoforms other than that which is recorded in thedatabases and thoroughly described in the bibliography.

The sequence corresponding to messenger RNA which contains the codingsequence of the porcine sst5 receptor, as well as the uncoded 5′(GI58223147) and 3′ regions, have recently been cloned (Duran et al.,2005; awaiting publication). During the cloning process of the porcinesst5 through the RACE PCR technique, partial, and then total sequencesof messenger RNA variants had been obtained through alternativeadjustment, which coded two new receptor isoforms, as was the case ofrat sst2, but in this case, they coded six and three transmembranedomains called porcine sst5B and sst5C isoforms (p-sst5B and p-sst5C),respectively.

There is data on truncated GPCRs produced by alternative adjustment ofmessenger RNA, which code proteins that form structures of less thanseven transmembrane domains, as has been previously described for GHRHreceptors (Rekasi et al., 2000), GnRH (Pawson et al., 2005),prostaglandin (Ishii et al., 2001), etc., some of which are functionalwith potential relevance in tumor processes.

According to the results obtained for porcine sst5, cloning of potentialhuman homologues of the porcine sst5B and sst5C isoforms was initiatedthrough RACE PCR, in order to subsequently evaluate their presence andsignificance in endocrine tumors through the PCR technique.

BIBLIOGRAPHY CITED IN THE TEXT

-   1. Moller L N, Stidsen C E, Hartmann B, Holst J J. 2003.    Somatostatin receptors. Biochemica et Biophysica Acta, 1616: 1-84.-   2. Castaño J P, Torronteras R, Ramírez J L, Gribouval A,    Sánchez-Hormigo A, Ruiz-Navarro A, Gracia-Navarro F. 1996.    Somatostatin increases growth hormone (GH) secretion in a    subpopulation of porcine somatotropes: evidence for functional and    morphological heterogeneity among porcine GH-producing cells.    Endocrinology, 137:129-136.-   3. Rekasi Z, Czompoly T, Schally A V, Halmos G. 2000. Isolation and    sequencing of cDNAs for splice variants of growth hormone-releasing    hormone receptors from human cancers. PNAS, 97: 10561-10566.-   4. Pawson A J, Maudsley S, Morgan K, Davidson L, Naor Z,    Millar R. 2005. Inhibition of human type I gonadotropin-releasing    hormone receptor (GnRHR-I) function by expression of a human type II    GnRHR gene fragment. Endocrinology. 146(6):2639-2649.-   5. Ishii Y, Sakamoto K. 2001. Suppression of protein kinase C    signaling by the novel isoform for bovine PGF2a Receptor1.    Biochemical and Biophysical Research Communications, 285: 1-8.-   6. Landa R L, Harbeck M, Kaihara K, Chepurny O, Kitiphongspattana K,    Graf O, Nikolaev V O, Lohse M J, Holz G G, Roe M W. 2005. Interplay    of Ca²⁺ and cAMP signaling in the insulin secreting MIN6 β-cell    line. Journal of Biological Chemistry, 2;280(35):31294-31302.-   7. Vilardaga J P, Bunemann M, Krasel C, Castro M & Lohse M J. 2003.    Measurement of the millisecond activation switch of G    protein-coupled receptors in living cells. Nat Biotechnol 21    807-812.

DESCRIPTION OF THE INVENTION

The following concepts are detailed for the proper interpretation of thepresent text: A “somatostatin receptor” is a transmembrane proteincoupled to a guanylate cyclase-type protein which belongs to the seventransmembrane domain type family of proteins, activated bysomatostatin-type hypothalamic peptides.

“RACE-PCR” refers to Random Amplification of cDNA Ends. It entails a PCR(Polymerase Chain Reaction) based technique through which known sequenceoligonucleotides are introduced within unknown sequences of cDNA, whichare used as target sequences for the amplification of the cDNA regionincluded between said oligonucleotides and the region of interestthrough PCR.

Hypophyseal Cushing's refers to “Cushing's Syndrome” or hypercorticism.It is a disease brought about by an increase in the production of thehormone cortisol or by the excessive use of this and other steroidhormones. Cushing's Syndrome is hypophyseal when it is due to anexcessive production of adrenocorticotropic hormone by the hypophysis orpituitary gland.

The present invention includes the determination of the DNA sequencethat codes two new isoforms of the human somatostatin receptor type 5(sst5), referred to as sst5B and sst5C, with five and four transmembranedomains, respectively, brought about by an alternate adjustment of themessenger RNA contained in the genomic sequence established in the database with access number GI13937340 (FIG. 1). Through the proceduredescribed in the mode of executing the invention, recombinant DNAmolecules are obtained which code polypeptides that exhibit thestructural motifs of somatostatin receptors, at least in part of thesequence. The invention also refers to polynucleotidic DNA sequenceswhich are hybridized with those of these new isoforms under restrictiveconditions, which entails a level of homology of a least 60% between itssequences of nucleotides, preferably with 75% homology and ideally with90% homology, or that they are derived from them through degeneration ofthe genetic code or by mutagenesis.

The procedure used in the invention allows functional recombinantpolypeptides to be obtained for later study. Recombinant DNA is insertedinto expression vectors in such a manner that they contain a sequence ofnucleotides such as those described in SEQ ID 1, SEQ ID 3, SEQ ID 5, andSEQ ID 7, or derived from them. Both polypeptides expressed inexpression vectors within the host cells provide a system ofscrutinizing new drugs and compounds capable of joining the sst5B andsst5C isoforms in vivo and in vitro in a selective manner, as well assystems of studying the modulation of second messenger routes for eachone of the isoforms in response to drugs.

As such, the invention that is the subject matter of this applicationrefers to a purified human nucleic acid characterized by coding anisoform of the human somatostatin receptor type 5 (sst5) selected fromamongst: sst5B (SEQ ID 5), sst5C (SEQ ID 7), its complementary sequence,and a sequence with at least 90% homology with the previous sequences,as well as fragments of the previous sequences.

Likewise, the invention also refers to a purified human nucleic acidwhich is characterized by including a partial coding sequence containedin SEQ ID 1 and SEQ ID 3.

In a specific embodiment, the invention refers to a human nucleic acidcharacterized by including the 3′RACE PCR fragment corresponding tosst5B, the sequence of which is SEQ ID 1, or fragments thereof. Inanother particular embodiment, the invention refers to a human nucleicacid characterized by including the 3′RACE PCR fragment corresponding tosst5C, the sequence of which is SEQ ID 3, or fragments thereof.

In a preferred embodiment, the invention refers to a purifiedpolypeptide characterized by its sequence of amino acids being definedin SEQ ID 2, SEQ ID 4, SEQ ID 6, and SEQ ID 8, which is coded by one ofthe oligonucleotides described previously in the text.

Moreover, the invention refers to an expression vector characterized byincluding the nucleotide sequence described previously, which istranscriptionally coupled to an exogenous promoter. In a specificembodiment, that expression vector is characterized by said nucleotidesequence coding a polypeptide as defined previously in this text.

In a specific embodiment, the methods described above are characterizedby being carried out in vitro. In a preferred embodiment, said search iscarried out on complete cells. In a preferred embodiment, said method ischaracterized by the polypeptides detailed in SEQ ID 2, SEQ ID 4, SEQ ID6, and SEQ ID 8 originating from an expression vector defined previouslyin the text. In a more preferred embodiment, said polypeptidecorresponds to one of those coded by SEQ ID 1, SEQ ID 3, SEQ ID 5, SEQID 7, or fragments thereof.

Moreover, the invention also refers to new pairs of oligonucleotidesdetailed in the sequences SEQ ID 9, SEQ ID 10, SEQ ID 11, SEQ ID 12, SEQID 13, and SEQ ID 14, or sequences homologous to them by at least 90%,which allow the sst5 human isoforms A, B, and C to be amplified throughPCR. In a specific embodiment, the invention refers to the use of saidoligonucleotides for the selective amplification of the sst5A, sst5B,and sst5C isoforms through any variant of PCR. In a preferredembodiment, the invention also refers to the use of saidoligonucleotides for the study of the quantitative tissular distributionof sst5A, sst5B, or sst5C in normal and tumoral tissues.

In another specific embodiment, the invention refers to a DNA copycharacterized by hybridizing with the total or partial sequencescontained in SEQ ID 1, SEQ ID 3, SEQ ID 5, or SEQ ID 7.

Moreover, the invention also refers to a DNA copy contained in SEQ ID 1,SEQ ID 3, SEQ ID 5, SEQ ID 7, or sequences homologous by at least 90%,characterized by being capable of silencing the gene expression of thesst5B and sst5C isoforms independently or jointly.

A specific embodiment of the present invention refers to the use of thesequences contained in SEQ ID 1, SEQ ID 3, SEQ ID 5, or SEQ ID 7 togenerate selective antibodies which discriminate between the sst5B andsst5C isoforms.

The present invention also allows for the development of new drugscapable of selectively joining the new isoforms of the somatostatinreceptor type 5, sst5B and sst5C, which act as agonists, antagonists, orinverse agonists by using second messenger measuring techniques such asmicro-fluorometric measurement of intracellular calcium (Landa et al.,2005). More specifically, the insertion of the recombinant DNA containedin SEQ ID 1, SEQ ID 3, SEQ ID 5, and SEQ ID 7, or their derivatives, inpCDNA3 (Invitrogen) type eucaryote expression vectors, allows for thetransfection of these recombinant structures in CHO-K1 or HEK-293T typetumoral cell lines, which are widely used for the study of othersomatostatin receptor subtypes. The process, the methodology of whichmay be seen in a more detailed manner in Landa et al., (Landa et al.,2005) may be schematized in a general manner as indicated below:

-   -   (1) Cultivation of the cell line to be transfected using sterile        glass slide covers.    -   (2) Transfection of the cellular line with the recombinant        plasmid of interest.    -   (3) Incubation of the transfected cells with 2.5 μM of Fura-2 AM        (Molecular Probes, Eugene) for 30 minutes at 37° C. in DMEM        medium supplemented with 20 mM of NaHCO₃ at 7.4 pH.    -   (4) Assembly of the slide covers which the cells are adhered to        in a camera coupled to an inverted microscope such as the Nikon        Eclipse TE 2000 E, coupled to a Hamamatsu CCD camera (Hamamatsu        Photonics, Hamamatsu), both of which are controlled with the        MetaMorph and MetaFluor (Molecular Devices) software.    -   (5) Analysis of the transfected cells with 40× oil immersion        objective through selective excitation to 340 and 380 nm and        measuring the output signal to 505 nm at 5 second intervals.    -   (6) The changes in intracellular Ca²⁺ concentration before and        after the administration of the drug in question are analyzed as        a quotient of the intensities of the image obtained with the        respective excitation wave lengths at 340 and 380 mm by using        the MetaFluor software.

The present invention also allows for the development of drugs whichalter the basal state of the new isoforms of the somatostatin receptortype 5, sst5B and sst5C. In this manner, this invention would allow FRET(Fluorescence Resonance Energy Transfer) technology to be used tomeasure the physical interaction of both sst5B and sst5C isoformsbetween themselves, as well as with other proteins that belong to theGPCR family. Through this technique, changes in the receptor's basalstate may be studied quickly and accurately, whether they entailaggregation or dissociation of ternary protein complexes in response toa drug. More specifically, the insertion of the recombinant DNAcontained in SEQ ID 1, SEQ ID 3, SEQ ID 5, and SEQ ID 7, or theirderivatives, in E-GFPN1 (Clontech) type eucaryote expression vectors,such as E-CFPN1 and E-YFPN1, would allow these recombinant structures tobe cotransfected in HEK-293AD type tumoral cell lines.

The process, the methodology of which may be seen in a more detailedmanner in Vilardaga et al., (Vilardaga et al., 2003) may be schematizedin a general manner as indicated below:

-   -   (1) Cultivation of the cell line to be transfected using sterile        glass slide covers.    -   (2) Cotransfection of the cell line with the recombinant        plasmids of interest.    -   (3) Assembly of the slide covers which the cells are adhered to        in a camera coupled to an inverted microscope such as the Nikon        Eclipse TE 2000 E, coupled to a Hamamatsu CCD camera (Hamamatsu        Photonics, Hamamatsu), both of which are controlled with the        MetaMorph and MetaFluor (Molecular Devices) software.    -   (4) Analysis of the transfected cells with 40× oil immersion        objective through selective excitation to 440 and 495 nm and        measuring the signal to 510 and 540 nm, respectively, at 5        second intervals.    -   (5) The changes in the intensity of the signal of both        fluorescent molecules before and after the administration of the        drug in question are analyzed as a quotient of the intensities        of the image obtained with the respective excitation wave        lengths at 510 and 540 nm by using the MetaFluor software.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Amplification drawing of partial sequences corresponding to thesst5B and sst5C isoforms through the 3′RACE PCR technique. The stagesdescribed in the text are presented and the oligonucleotides used ineach case are displayed according to the nomenclature of Table 1.

FIG. 2: Amplification drawing of coding sequences of the sst5B and sst5Cisoforms through the PCR amplification technique and triple ligation.The stages described in the text are presented and the oligonucleotidesused in each case are displayed according to the nomenclature of Table1.

MODES OF EXECUTING THE INVENTION

An example is described below for greater illustration of the invention,but does not limit it in any way.

EXAMPLE

The following stages described in FIG. 1 were followed to clone partialsequences of the sst5B and sst5C isoforms:

-   -   (1) Total isolation and retrotranscription of RNA of various        tumoral tissues.    -   (2) Amplification of 3′ region of the sst5B and sst5C isoforms        through RACE PCR; and    -   (3) reamplification using oligonucleotides nested in those used        in step (2).    -   (4) Cloning the PCR products from step (3) and sequencing to        determine their proper sequence.    -   (5) Verification of the initiation of transcription of the sst5B        and sst5C isoforms through PCR using the DNA copy created in (1)        as a mold.    -   (6) Reamplification of (5) in order to verify the specificity of        the PCR bands obtained in that step.

The described amplification method was carried out by following theindications of the Invitrogen® Life Technologies “GeneRacer® Kit”,except for steps (5) and (6).

The strategy schematized in FIG. 2 was used to clone the codingsequences and functional expression of the sst5B and sst5C isoforms:

-   -   (1) Both exon 1 (E1) and exon 2 (E2) corresponding to each of        the isoforms were amplified from human genomic DNA.    -   (2) Enzymatic digestion of the PCR fragments, and    -   (3) Mutual ligation of exons E1 and E2 within an expression        vector (not shown in the drawing).

In addition, pairs of oligonucleotides were designed capable ofdiscriminating each of the sst5A, sst5B, and sst5C isoforms (SEQ ID 9 toSEQ ID 14) and which can be used for quantitative purposes, selectivelydiscriminating each isoform under the specific PCR conditions detailedfurther along in the text.

Isolation of Nucleic Acids.

Isolation of RNA. This was carried out by using the reagent Trizol fromInvitrogen® according to the recommendations indicated by that company.Tissues from two hypophyseal adenomas diagnosed as non-functioning and asample of hypophyseal “Cushing's” were used as starting material for thetotal isolation of RNA designated for cloning. The resulting RNA wasresuspended in a final volume of 12 μl of DEPC-treated H₂O, of which 1μl was used for spectrophotometric quantification. 2 μg of RNA from eachof the tumors was used for retrotranscription in a final volume of 20μl. RNA from the HeLa tumoral cell line from the “GeneRacer kit” wasalso used, and the same quantity of RNA was used for theretrotranscription reaction (FIG. 1.1). The indications from theInvitrogen® “GeneRacer® kit” were followed for the retrotranscriptionreaction. A heterogeneous battery of hypophyseal tumoral tissues wasalso used for diagnostic purposes, from which a quantity of between 15and 100 mg was used for the extraction of total RNA. The resulting RNAwas also resuspended in a final volume of 12 μl of DEPC-treated H₂O, ofwhich 1 μl was used for spectrophotometric quantification. Between 2 and5 μg of RNA from each of the tumors, according to the yield of eachextraction, was used for retrotranscription. The retrotranscriptionreaction was carried out in a total volume of 20 μl using the BDBiosciences “PowerScript” enzyme according to the manufacturer'sindications.

Isolation of DNA. This was carried out using the reagent Trizol fromInvitrogen®, using 10⁷ human lymphocytes as the starting material. Thegenomic DNA obtained was quantified spectrophotometrically.

Amplification by PCR and Obtaining Partial Sequences of the sst5B andsst5C Isoforms.

As indicated previously, the “GeneRacer® Kit” commercial amplificationsystem from Invitrogen® was used in combination with the specificallydesigned oligonucleotides indicated in Table 1.

TABLE 1Oligonucleotides used for selective amplification of partial sequencesof h-sst5B and h-sst5C. 5′ → 3′ sequence, position in  Namesequence of reference and  Reading (SEQ ID) amino acids sequencedirection Reference Hum_sst5_ATG  1-ATGGAGCCCCTGTTCCCAGCCT-22 SenseGI39756975 (15) 1-MEPLFPA-7 Ra_hum_sst5_3′490-TGGGTCCTGTCTCTGTGCATGTC-512 Sense GI39756975 (16) 164-WVLSLCM-170Ra_hum_sst5_3′N  523-CTGGTGTTCGCGGACGTGCAG-543 Sense GI39756975 (17)175-LVFADVQ-181 sst5B-C_E1_U_ 1- Sense GI39756975 HindIII (18)TCAAGCTTCGATGGAGCCCCTGTTCCCAGC- 20 1-MEPLFP-6 sst5B-E1_599-CGGCGCGAAGAAGCCCAGCAC-619 Anti- GI39756975 L_blunt (19)207-VLGFFAP-213 sense sst5B-E2-U_blunt 2275-CTGCTGAGAGGCAGCGGCC-2293Sense GI13937340 (20) LLRGSG sst5B-E2-L_BamHI 2437- Anti- GI13937340(21) TTAGGATCCTCAGAGCAAGGCCAAGTTGCC- sense 2457 GNLALL sst5C-E1_675-GTTGCAGGTACCGCCCTCCTG-695 Anti- GI39756975 L_blunt (22)181-QEGGTCN-187 sense sst5C-E2_ 2548-CGTCTGCCCAGAGCAGGACCTC-2569 SenseGI13937340 U_blunt (23) RLPRAGP sst5C-E2_L_BamHI 2617- Sense GI13937340(24) ACTGGATCCTCAGCCTGGGCCTTTCTCCTG- 2637 QEKGPG *The bases appearing initalics represent restriction enzyme targets.

After the retrotranscription reaction, 100 ng of DNA copy was used foreach PCR reaction, using the Ra_hum_sst5_(—)3′ (SEQ ID 16) and GeneRacer3′ oligonucleotides (FIG. 1.3), in which the samples underwent aninitial 2 minute denaturation at 94° C., followed by five 30 secondrepetitions of denaturation at 94° C. and 1 minute 30 seconds ofalignment and extension at 72° C., in addition to another thirty-fivecycles equal to the previous one but using the less astringent alignmenttemperature of 66° C. The amplification program continued with 7 minutesof extension at 72° C. in order to finalize the synthesis of incompletePCR products. 1 μl from each PCR product was reamplified with theRa_hum_sst5_(—)3′N (SEQ ID 17) and GeneRacer 3′N nested oligonucleotides(FIG. 1.4), with the samples undergoing an initial 2 minute denaturationat 94° C., and then thirty-two 30 second denaturation cycles at 94° C.,30 seconds of alignment at 66° C., and 40 seconds of extension at 72° C.The amplification program continued with 7 minutes of extension at 72°C. in order to finalize the synthesis of incomplete PCR products. Amixture of Biotools Certamp polymerases provided with a plugspecifically for complex mixtures was used for both amplifications. Thedifferent PCR reactions were carried out with all DNA copiesconcurrently. The PCR products were observed in 1% agarose gel and thebands obtained were purified by using the QuiaQuick Mini Elute (Quiagen)commercial kit. The PCR products purified in this manner were clonedwith blunt ends in the EcoRV restriction site of the pBluescript KSII+vector and were sequenced, resulting in the sequence corresponding to609 pairs of bases (SEQ ID 1) obtained from the DNA copy of HeLa cellsand another sequence of 197 base pairs (SEQ ID 3) obtained from the DNAcopy from the hypophyseal “Cushing's”. Both DNA copies obtained from theHeLa cells and the hypophyseal “Cushing's” were then amplified with theHum_sst5_ATG (SEQ ID 15) and GeneRacer 3′ oligonucleotides (FIG. 1.5)and the products of those reactions were reamplified with the sameHum_sst5_ATG oligonucleotide and the GeneRacer 3′N nestedoligonucleotide (FIG. 1.6). The same program was used for both PCRamplification reactions consisting of an initial 2 minute denaturationat 94° C. followed by forty 30 second denaturation cycles at 94° C., 30seconds of alignment at 62° C., and 1 minute 30 seconds of extension at72° C. Observation in 1% agarose gel allowed the existence of a band ofaround 1 kilobase obtained from the DNA copy of HeLa, and another bandof around 700 base pairs obtained from the DNA copy of the hypophyseal“Cushing's”, to be proven. This result allowed it to be proven that,analogously to the homologous porcine sequences, both isoforms, referredto as sst5B and sst5C, respectively, share a putative beginning from thecommon translation with the sst5A isoform, the sequence of which waspreviously known and available in the databases with access numberGI39756975.

Obtaining the Coding Sequences Corresponding to the sst5B and sst5CIsoforms.

A strategy based on the independent amplification of the two exons thatcomprise each isoform (FIG. 2), as well as the subsequent ligation ofboth fragments within a eucaryote expression vector, was followed forthe cloning and functional expression of the sst5B and sst5C isoforms.More specifically, starting from genomic DNA as a mold, exon 1 (E1) ineach isoform was amplified through PCR, using a common senseoligonucleotide for sst5B and sst5C, sst5B-C_E1_U_HindIII (SEQ ID 18),which incorporates a sequence of restriction for the HindIII enzyme, andan antisense oligonucleotide specific to each isoform, sst5B-E1_L_blunt(SEQ ID 19) and sst5C-E1_L_blunt (SEQ ID 22) for sst5B and sst5C,respectively (FIGS. 2.1 and 3.2). Exons 2 (E2) were amplified in ananalogous manner, with the specific pairs of sense and antisenseoligonucleotides sst5B-E2-U_blunt (SEQ ID 20)/sst5B-E2-L_BamHI (SEQ ID21) for isoform sst5B and sst5C-E2_U_blunt (SEQ ID 23)/sst5C-E2_L_BamHI(SEQ ID 24) for isoform sst5C. The four PCR reactions were carried outconcurrently using a program consisting of an initial 2 minutedenaturation at 94° C. followed by thirty-four 30 second denaturationcycles at 94° C., 30 seconds of alignment at 62° C., and 40 seconds ofextension at 72° C. In every case, a high-fidelity polymerase was usedwith a Pfu Ultra (Stratagene) type copy supplementing the reactions with1M betaine (Sigma). Through these PCR reactions, a sequence ofrestriction for the HindIII enzyme was able to be incorporated into the5′ end of each E1, the 3′ blunt end of the E1, the 5′ blunt end of theE2, and a sequence of restriction for the BamHI enzyme in the 3′ end ofeach E2. It was previously verified that none of the enzymes ofrestriction had additional cut sites within the sequences of interest.The PCR fragments obtained were purified by using the QuiaQuick MiniElute (Quiagen) commercial kit, and after enzymatic digestion withHindIII and BamHI, they were linked through a triple reaction within thepCDNA3+ (Invitrogen) eucaryote expression vector which had beenlinearized with the previous enzymes of restriction. Both sst5B-pCDNA3+and sst5C-pCDNA3+ structures were sequenced at least in duplicate inorder to verify the integrity of the sequences and to compare them withthe GI13937340 genomic sequence, which contains the two isoforms, byusing the BLAST 2 SEQUENCES programhttp://www.ncbi.nlm.nih.gov/blast/b12seq/wblast2.cgi.

Selective Differential Amplification of Partial Sequences Correspondingto the sst5A, sst5B, and sst5C Isoforms for Qualitative Purposes.

Pairs of oligonucleotides were developed for diagnostic purposes, whichallow selective discrimination by PCR of each of the human sst5, sst5A(GI39756975), sst5B, and sst5C isoforms. The pair of oligonucleotidesHum_sst5A_cuant_U/Hum_sst5A_cuant_L, SEQ ID 9 and SEQ ID 10,respectively, amplify a PCR product of 154 base pairs using an alignmenttemperature of 68° C. The pair of oligonucleotides Hum_sst5B_cuant_U /Hum_sst5B_cuant_L, SEQ ID 11 and SEQ ID 12, respectively, amplify a PCRfragment of 142 base pairs, using an alignment temperature of 68° C.,contained in the sequence corresponding to sst5B (SEQ ID 5) and does notamplify the sst5C or sst5A (GI39756975) isoforms, while it produces anamplification product of 1643 base pairs contained in the GI13937340human genomic sequence which includes the intron situated between exonsE1 and E2 of the sst5B isoform. The pair of oligonucleotidesHum_sst5C_cuant_U/Hum_sst5C_cuant_L, SEQ ID 13 and SEQ ID 14,respectively, amplify a PCR fragment of 137 base pairs using analignment temperature of 68° C., contained in the sequence correspondingto sst5C (SEQ ID 6) and also amplify a fragment of 488 base pairscontained in the sequence corresponding to the sst5B and anotherfragment of 1989 base pairs contained in the human genomic sequenceGI13937340, which includes the intron situated between exons E1 and E2of the isoform sst5C. The three PCR reactions are carried outconcurrently using a common PCR program consisting of an initial 2minute denaturation at 94° C. and 37 10 second repetitions at 94° C., 10seconds of alignment at 68° C. and 10 seconds of extension at 72° C. Byusing these PCR conditions, each pair of oligonucleotides only amplifiesthe specific PCR fragment of each isoform selectively, withoutamplifying the other additional sequences mentioned previously. In everycase, the reactions were supplemented with 1M betaine (Sigma). Thismethodology allowed the selective presence of the sst5B isoform to beobserved in various hypophyseal tumors clinically classified asnon-functioning.

1. A method of selectively amplifying a human somatostatin receptor type5 nucleic acid, wherein purified oligonucleotides, fragments, homologs,or complementary nucleic acids thereof comprising a sequence having atleast 90% homology with SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13 or SEQ ID NO:14, are used for the PCR amplificationof a human somatostatin receptor type 5 nucleic acid.
 2. A method ofgene silencing the expression of either or both sst5B or sst5Csomatostatin receptor type 5 genes, said method comprising administeringa somatostatin receptor type 5 nucleic acid, fragment, homolog orcomplementary nucleic acid comprising a sequence having at least 90%homology to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7.